This study aimed to determine the protective effect of cashew nut-derived protein hydrolysate with high dietary fiber (AO) in cerebral ischemic rats induced by the occlusion of right middle cerebral artery (Rt.MCAO). Acute toxicity was determined and data showed that LD50 of AO > 5000 mg/kg BW. To determine the cerebroprotective effect of AO, male Wistar rats were orally given AO at doses of 2, 10, and 50 mg/kg for 14 days and subjected to Rt.MCAO. Brain infarction volume, neurological score, spatial memory, serum lipid profiles, and C-reactive protein together with the brain oxidative stress status were assessed. All doses of AO significantly decreased brain infarction in cortex, hippocampus, and striatum together with the decreased oxidative stress status. The improvement of spatial memory and serum C-reactive protein were also observed in MCAO rats which received AO at all doses. In addition, the decreased serum cholesterol, TG, and LDL but increased HDL were observed in MCAO rats which received high dose of AO. Taken all together, AO is the potential protectant against cerebral ischemia. The improvement of oxidative stress, inflammation, and dyslipidemia might play roles in the actions. However, further researches are required to understand the precise underlying mechanism.
Currently, ischemic stroke has been regarded as an important cause of mortality and morbidity at all economic levels worldwide [
Recently, nutrition has been recognized as an important factor for stroke prevention [
Cashew or
Cashew nuts were cleaned in order to remove all foreign matters such as dust, stone, and dirt, cut into small pieces, and soaked in the boiling water for 10 minutes. Then, distilled water was added at a ratio of 1 : 1 (w/v) and adjusted to pH 8. The suspension was stirred and boiled in water bath at 55°C for 30 minutes. The sample was hydrolyzed by adding 2% alcalase enzyme. After 4 hours of hydrolysis, the inactivation process of enzyme was induced by heating at 90°C for 15 minutes in water bath. Then, the mixture was subjected to a 4100-rpm centrifugation for 10 minutes. The supernatant was collected and concentrated by using freeze dryer [
In this study, bagasse of cashew was obtained from the waste generated by fruit pulp industrialization and provided by Srisuphaluck Orchid Company, Phuket, Thailand. Cashew apple residue was chopped into small pieces and dissolved in water at a ratio 1 : 1 (w/v). The suspension was adjusted to pH 5, boiled in water bath at 60°C for 10 minutes, and filtered through cheese cloth. The filtrate was immersed in 95% ethanol at room temperature for 24 hours. The mixture was occasionally agitated. This process was repeated twice. Finally, it was dried in oven at 65°C and grinded in to powder and used as a sample of dietary fiber [
To obtain the desired doses of the cashew nut derived protein hydrolysate with high fiber (AO), the protein hydrolysate at doses of 1, 10, and 100 mg/kg BW were mixed with dietary fiber at dose of 2 g/kg BW.
DPPH radical scavenging activity was measured using the method of Cotelle et al. [
Reducing power of cashew hydrolysate was performed according to method of Benzie and Strain [
COX-2 activity was measured using the method of Jang et al. [
Healthy male and female Wistar rats (300–350 g) from the National Laboratory Animal Center, Salaya, Nakhon Pathom, were used as experimental animals. They were randomly housed 5 per cage, maintained in 12 : 12 light : dark cycle, and given access to food and water ad libitum. The experiments were strictly performed in accordance with the internationally accepted principles for laboratory use and care of the European Community (EEC directive of 1986; 86/609/EEC). The experiment protocols were approved by the Institutional Animal Care and Unit Committee Khon Kaen University, Thailand (Record no. AEKKU 29/2015). All operations were performed under the pentobarbital sodium anesthesia in order to minimize animal suffering.
Acute toxicity was determined according to the protocols described in OECD Guideline 423. In brief, a total of 20 rats (male 10, female 10) were randomly divided into control and treatment groups. The treatment group was orally given the cashew nut-derived protein hydrolysate with high fiber at a single dose of 5000 mg/kg BW while control group received vehicle. The general appearance and behavioral changes were recorded at 1, 2, 4, and 6 hour-observation period after the daily administration for 14 days. All rats were weighed and we observed mortality, behavioral pattern (salivation, fur, lethargy, and sleep), changes in physical appearance, injury, pain, and signs of illness once daily during the 14-day study period. At the end of study, the blood was collected under anesthesia and the hematological and clinical chemistry changes were determined by Srinagarind hospital. After the collection, the principal organs were excised, weighed, and examined both at macroscopic and at microscopic levels. The evaluation at microscopic level was performed via histological study.
In order to determine the cerebroprotective effect of cashew nut-derived protein hydrolysate with high dietary fiber, male Wistar rats (300–350 g) were randomly divided into 7 groups as follows.
The animals in groups II–VII were orally given the assigned substances at a period of 14 days and subjected to the occlusion of right middle cerebral artery (Rt.MCAO), whereas animals in group I were treated with vehicle at the same period and exposed to sham operation. All treated substances were continually administered throughout a 21-day study period. The biochemical assays were performed at the end of study.
12 hours prior to the surgery, animals were exposed to food deprivation but were allowed to access water. Then, they were anesthetized by the intraperitoneal injection of thiopental sodium at dose of 60 mg/kg BW. After the anesthetization, the occlusion of right middle cerebral artery was induced in all rats (MCAO) as previously described [
All rats were killed 24 hours after MCAO, and the brain was removed and sectioned at 2-mm thickness. Sections were immersed in 2% TTC (2,3,5-triphenyl tetrazolium chloride) for 30 minutes at 37°C. The staining brain sections were photographed, and the infarction area was determined by measuring the white area of brain section with computer software (Image J 1.4 V).
The neurological score was evaluated according to the method of Bederson and coworkers [
Spatial memory was evaluated by using Morris water maze test [
At the end of experiment, homogenate of right hippocampus, cerebral cortex, and striatum were prepared in 1 mL of 0.1 M phosphate buffer, pH 7.4. The obtained brain homogenate was adjusted to 10% w/v and subjected to a 3,000
Level of malondialdehyde (MDA) was monitored by using thiobarbituric acid reacting substances (TBARS) assay. In brief, 100
The determination of superoxide dismutase (SOD) was carried out based on the inhibitory effect of SOD on the reduction of nitroblue tetrazolium (NBT) by the superoxide anion generated by the system xanthine/xanthine oxidase as previously described elsewhere [
Serum C-reactive protein was determined according to the protocol provided by Thermo Scientific with slight modifications. In brief, 100
Serum preparation was prepared after blood collection. The separation of serum was performed by using cooling centrifugation at 2500 rounds per minute (rpm) for 10 minutes. All serum hematological and clinical chemistry changes were determined using photometric analyzer at Srinagarind hospital, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Data are shown as mean ± standard error of mean (SEM). Statistical significance was set at
The biological activities related to the pathophysiology of cerebral ischemia were assessed to evaluate the cerebroprotective potential of the developed product. It was found that the IC50 value of the developed product obtained via DPPH was less than 5
The amino acids composition of cashew nut-derived protein hydrolysate in this study was shown in Table
Amino acid compositions from cashew nut-derived protein hydrolysate.
Amino acid profiles (mg/100 g extract) | Cashew nut |
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Alanine | 918 |
Arginine | <5.00 |
Aspartic acid | 1266 |
Cystine | 632 |
Glutamic acid | 3055 |
Glycine | 784 |
Histidine | 1823 |
Hydroxylysine | <5.00 |
Hydroxyproline | 51 |
Isoleucine | 874 |
Leucine | 2229 |
Lysine | 5287 |
Methionine | 387 |
Phenylalanine | 2139 |
Proline | 1011 |
Serine | 674 |
Threonine | 414 |
Tryptophan | 107 |
Tyrosine | 1064 |
Valine | 1347 |
Our data obtained from the amino acid analysis suggested that a cashew nut-derived protein hydrolysate was a high quality food for feeding because it contained abundance of essential amino acids. In addition it also possessed both antioxidant and anti-inflammation activities.
Our study showed that no abnormal clinical signs, behavioral changes, body weight changes, macroscopic findings, or organ weight changes were observed. It was found that all animals used in this study survived throughout a 14-day study period. The body weight data were shown in Table
Body weight of rats during a 14-day observation period, after a single administration of cashew nut-derived protein hydrolysate with high fiber at dose of 5,000 mg/kg BW via oral route. (
Group | Body weight (g) | Percent change of body weight | |
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Day 1 | Day 14 | Day 14 | |
Male | |||
Control |
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AO 5,000 mg/kg BW |
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Female | |||
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AO 5,000 mg/kg BW |
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Daily food and water intakes of rats during a 14-day observation period, after a single administration of cashew nut-derived protein hydrolysate with high fiber at dose of 5,000 mg/kg BW via oral route. (
Group | Food intake (g/rat/day) | Water intake (ml/rat/day) | ||
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Day-1 | Day-14 | Day-1 | Day-14 | |
Male | ||||
Control |
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AO 5000 mg/kg BW |
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Female | ||||
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AO 5000 mg/kg BW |
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The weight of various organs of male and female rats after a single administration of a cashew nut-derived protein hydrolysate with high fiber at dose of 5,000 mg/kg BW via oral route. (
Visceral organ (g/kg BW) | Male | Female | ||
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Control | AO 5000 mg/kg BW | Control | AO 5000 mg/kg BW | |
Brain |
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lung |
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liver |
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Heart |
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Spleen |
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Pancreas |
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Stomach |
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Intestine |
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Thymus gland |
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Urinary bladder |
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Kidney | ||||
Left |
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Right |
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Adrenal gland | ||||
Left |
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Right |
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Salivary gland | ||||
Left |
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Right |
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Testis/ovary | ||||
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Right |
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Hematological parameters of experimental rats after a 14-day observation period following a single administration of cashew nut-derived protein hydrolysate with high fiber at dose of 5,000 mg/kg BW. (
Parameter | Male | Female | ||
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Control | AO 5000 mg/kg BW | Control | AO 5000 mg/kg BW | |
Red blood cell (106/ul) |
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Hemoglobin (g/dL) |
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Hematocrit (%) |
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White blood cells (103/ul) |
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Platelet count (103/ul) |
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Neutrophils (%) |
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Lymphocytes (%) |
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Monocytes (%) |
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Eosinophil (%) |
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Basophil (%) |
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Mean corpuscular volume (fL) |
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Mean corpuscular Hemoglobin (pg) |
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Mean corpuscular Hemoglobin concentration (g/dL) |
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Red blood cell distribution width (%) |
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Clinical chemistry parameters of experimental rats after a 14-day observation period following a single administration of cashew nut-derived protein hydrolysate with high fiber at dose of 5,000 mg/kg BW. (
Parameter | Male | Female | ||
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Control | AO 5000 mg/kg BW | Control | AO 5000 mg/kg BW | |
Glucose (mg/dl) |
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Cholesterol (mg/dl) |
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Triglyceride (mg/dl) |
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BUN (mg/dl) |
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Creatinine (mg/dl) |
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ALT (U/L) |
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AST (U/L) |
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ALP (U/L) |
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Total Bilirubin (mg/dl) |
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Sodium (mEq/L) |
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Potassium (mEq/L) |
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Chloride (mEq/L) |
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Bicarbonate (mEq/L) |
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Figure
Effect of cashew nut-derived protein hydrolysate with high fiber on brain infarct volume in hippocampus, cerebral cortex, and striatum. (a) Representative photographs of brain infarct volume in various groups assessed by using TTC staining. (b) Volume of brain infarct area, both core and penumbra area of various groups (
The effect of cashew-derived protein hydrolysate with high fiber on neurological score was shown in Figure
Effect of cashew nut-derived protein hydrolysate with high fiber on neurological score. (
Figure
Effect of cashew nut-derived protein hydrolysate with high fiber on spatial memory (a) effect on escape latency, (b) effect on retention time. (
The effect of the developed product on retention time was also explored and the results were shown in Figure
Figure
Effect of cashew nut-derived protein hydrolysate with high fiber on malondialdehyde (MDA) level in hippocampus, cerebral cortex and striatum. (
Effect of cashew nut-derived protein hydrolysate with high fiber on superoxide dismutase (SOD) activity in hippocampus, cerebral cortex and striatum. (
Effect of cashew nut-derived protein hydrolysate with high fiber on catalase (CAT) activity in hippocampus, cerebral cortex and striatum. (
Effect of cashew nut-derived protein hydrolysate with high fiber on glutathione peroxidase (GSH-Px) activity in hippocampus, cerebral cortex, and striatum. (
Since inflammation plays an essential role on the pathophysiology of cerebral ischemia, we also assessed the effect of the developed product on inflammation by using serum C-reactive protein as index and data were shown in Figure
Effect of cashew nut-derived protein hydrolysate with high fiber on serum C-reactive protein level. (
Table
Effect of various doses of cashew nut-derived protein hydrolysate with high fiber on lipid profiles. (
Group | Parameter | |||
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Cholesterol (mg/dL) | Triglyceride (mg/dL) | LDL (mg/dL) | HDL (mg/dL) | |
Vehicle + Sham operation |
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Vehicle + MCAO |
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Piracetam 250 mg/kg BW + MCAO |
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Vitamin C 250 mg/kg BW + MCAO |
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AO 2 mg/kg BW + MCAO + MCAO |
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AO 10 mg/kg BW + MCAO + MCAO |
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AO 50 mg/kg BW + MCAO + MCAO |
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The present data have demonstrated that single oral administration of cashew hydrolysate with high fiber (AO) at dose of 5000 mg/kg BW produced no effect on mortality, clinical signs, body weight, and organ weight both in male and in female rats throughout the 14 days of study period. Therefore, the cashew hydrolysate with high fiber should be classified in category 5 which is recognized as the lowest toxicity class according to the Guidance Document on Acute Oral Toxicity Testing based on oral LD50 value which were recommended by Organization for Economic Cooperation and Development [
Our in vitro data have clearly demonstrated that AO shows antioxidant and anti-inflammation activities. These effects are also confirmed by our in vivo data which show that AO at all doses used in this study enhance the activities of the main scavenger enzymes including SOD, CAT, and GSH-Px. The elevations of SOD and GSH-Px activities are observed in cerebral cortex, hippocampus, and striatum whereas the enhanced CAT is observed only in hippocampus and striatum. This pointed out CAT activity in cerebral cortex was less sensitive to the treatment. This was in agreement with the previous study which demonstrated that CAT in frontal cortex was less sensitive to pilocarpine treatment [
The current data also clearly demonstrated that the cashew nut-derived protein hydrolysate with high fiber also improved neurological score and spatial memory. Based on the previous information that the improved brain infarction was associated with the improvement of neurological score [
In this study, we also demonstrated that Rt.MCAO rats also showed the elevation of cholesterol. Interestingly, high dose of AO can decrease cholesterol, LDL, and TG but enhance HDL. Since dyslipidemia plays a vital role in the induction of endothelial dysfunction [
Interestingly, our data showed that AO at all dosage range used in this study showed the protective effect against cerebral ischemia induced by Rt.MACO better than the positive control or piracetam and vitamin C especially in cerebral cortex and striatum. Moreover, AO also improves neurological score, spatial memory, and dyslipidemia better than both substances mentioned earlier. Moreover, it is less toxic. Therefore it is worth further exploring the subchronic toxicity and precise underlying mechanism.
This study has clearly demonstrated that cashew nut-derived protein hydrolysate with high fiber (AO) is the potential cerebroprotectant against focal cerebral ischemia. The consumption safety is up to 5000 mg/kg BW. Therefore, it is practically safe. Since it can exert the effect on multitargets simultaneously, it may provide high benefit for the complex disorders such as stroke. However, further researches concerning subchronic toxicity and precise underlying mechanisms are required before moving forward to clinical study.
The founding sponsors had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results.
The authors declare no conflicts of interest.
This study was supported by Integrative Complementary Alternative Medicine Research and Development Center, Faculty of Medicine, Khon Kaen University, Thailand, Office of Higher Education Commission and Srisuphaluck Orchid Company through Public-Private Joint Research and Development Project.